The initiation of DNA replication in bacteria and mammalian cells is being studied. In bacteria, attention is focused on the inactivation of the complex of replication proteins. The stability of this complex is determined by one or two genes. Mutants affecting the stability will be isolated and used to dissect the components of the stabilizing system. The role of RNA in initiating replication is being studied in an in vitro replication system. An assay is proposed to detect initiation in vitro. The bacterial membrane has been altered by substituting specific unsaturated fatty acids for the ones usually incorporated. The effect of these altered membranes on ts DNA mutations will be studied. Mutants conditionally deficient (ts) in DNA replication will be isolated from mouse and human cells. These will be characterized for their ability to complement each other and for their physiological characteristics (divided into four suggested types). The ability of specific fusion hybrids to survive will be used as a tool to trace replication defects within single chromosomes.